The invention relates to novel 2-heteroarylcarboxamides, processes for their preparation, and their use for producing medicaments for the treatment and/or prophylaxis of diseases and for improving perception, concentration, learning and/or memory.
Nicotinic acetylcholine receptors (nAChR) form a large family of ion channels which are activated by the messenger acetylcholine which is produced in the body (Galzi and Changeux, Neuropharmacol. 1995, 34, 563-582). A functional nAChR consists of five subunits which may be different (certain combinations of α1-9 and β1-4,γ,δ,ε subunits) or identical (α7-9). This leads to the formation of a diversity of subtypes which differ in the distribution in the muscles, the nervous system and other organs (McGehee and Role, Annu. Rev. Physiol. 1995, 57, 521-546). Activation of nAChR leads to influx of cations into the cell and to stimulation of nerve cells or muscle cells. Selective activation of individual nAChR subtypes restricts this stimulation to the cell types which have a corresponding subtype and is thus able to avoid unwanted side effects such as, for example, stimulation of nAChR in the muscles. Clinical experiments with nicotine and experiments in various animal models indicate that central nicotinic acetylcholine receptors are involved in learning and memory processes (e.g. Rezvani and Levin, Biol. Psychiatry 2001, 49, 258-267). Nicotinic acetylcholine receptors of the alpha7 subtype (α7 nAChR) have a particularly high concentration in regions of the brain which are important for learning and memory, such as the hippocampus and the cerebral cortex (Séguéla et al., J. Neurosci. 1993, 13, 596-604). The α7 nAChR has a particularly high permeability for calcium ions, increases glutamatergic neurotransmission, influences the growth of axons and, in this way, modulates neuronal plasticity (Broide and Leslie, Mol. NeurobioL 1999, 20, 1-16).
Certain N-(1-azabicyclo[2.2.2]oct-3-yl)heteroaryl carboxamides for the treatment of, inter alia, psychoses are described in DE-A 37 24 059.
N-(Azabicycloalkyl)heteroaryl carboxamides, in particular N-(1-azabicyclo-[2.2.2]oct-4-yl)benzothiophene-3-carboxamides, are disclosed in WO 93/15073 and in EP-A 0 485 962 as intermediates for the synthesis of pharmaceutically active compounds.
1-Azabicycloalkanes and their action on the nicotinic α7-receptor are known from JP 14030084A.
U.S. Pat. No. 4,605,652 and EP-A 0 372 335 disclose, for example, N-(1-azabicyclo[2.2.2]oct-3-yl)thiophene-2-carboxamide and its memory-improving effect.
The present invention relates to compounds of the formula (I)
in which    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen or C1-C6-alkyl,    R3 represents hydrogen, halogen or C1-C6-alkyl,    A represents oxygen or sulfur,and    the ring B represents benzo, pyrido, pyrimido, pyridazo or pyridazino which are optionally substituted by radicals selected from the group consisting of hydrogen, halogen, C1-C6-alkanoyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, C1-C6-acylamino, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-alkylthio, C1-C6-alkylamino, heteroarylcarbonylamino, arylcarbonylamino, C1-C4-alkylsulfonylamino, di-(C1-C4-alkylsulfonyl)amino, arylsulfonylamino, di(arylsulfonyl)amino, C3-C6-cycloalkylcarbonylmethyl, 1,3-dioxapropane-1,3-diyl, amino(hydroxyimino)methyl and benzo,and their salts, solvates and solvates of the salts.
The compounds according to the invention can exist in stereoisomeric forms which are either like image and mirror image (enantiomers) or which are not like image and mirror image (diastereomers). The invention relates both to the enantiomers and diastereomers and to their respective mixtures. These mixtures of the enantiomers and diastereomers can be separated in a known manner into the stereoisomeric uniform components.
Compounds according to the invention can also be present in the form of their salts, solvates or solvates of the salts.
Salts which are preferred for the purposes of the invention are physiologically acceptable salts of the compounds of the invention.
Physiologically acceptable salts of the compounds according to the invention may be acid addition salts of the compounds with mineral acids, carboxylic acids or sulfonic acids. Particularly preferred examples are salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.
However, salts which may be mentioned are also salts with conventional bases, such as, for example, alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. calcium or magnesium salts) or ammonium salts derived from ammonia or organic amines such as, for example, diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabiethylamine, 1-ephenamine or N-methylpiperidine.
Solvates is the term used for the purposes of the invention for those forms of the compounds which form a complex with solvent molecules by coordination in the solid or liquid state. Hydrates are a special form of solvates in which the coordination takes place with water.
For the purposes of the present invention, the substituents generally have the following meaning:
C1-C6- and C1-C4-Alkoxy are a straight-chain or branched alkoxy radical having 1 to 6 and 1 to 4 carbon atoms, respectively. Preference is given to a straight-chain or branched alkoxy radical having 1 to 4, particularly preferably 1 to 3, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy.C1-C6- and C1-C4-Alkyl are a straight-chain or branched alkyl radical having 1 to 6 and 1 to 4 carbon atoms, respectively. Preference is given to a straight-chain or branched alkyl radical having 1 to 4, particularly preferably 1 to 3, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: methyl, ethyl, n-propyl, isopropyl, tert-butyl, n-pentyl and n-hexyl.(C1-C6)-Alkanoyl is a straight-chain or branched alkyl radical having 1 to 6 carbon atoms which carries a doubly attached oxygen atom in the 1-position and is attached via the 1-position. Preference is given to a straight-chain or branched alkanoyl radical having 1 to 4, particularly preferably 1 to 2, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: formyl, acetyl, propionyl, n-butyryl, isobutyryl, pivaloyl and n-hexanoyl.C1-C6-Alkylamino is a straight-chain or branched alkylamino radical having 1 to 6 carbon atoms, preferably 1 to 4, particularly preferably 1 to 3, carbon atoms. Nonlimiting examples include methylamino, ethylamino, n-propylamino, isopropylamino and tert-butylamino.(C1-C6)-Acylamino is an amino group having a straight-chain or branched alkanoyl substituent which has 1 to 6 carbon atoms and is attached via the carbonyl group. Preference is given to an acylamino radical having 1 to 4, particularly preferably 1 to 2, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: formamido, acetamido, propionamido, n-butyramido and pivaloylamido.C1-C4-Alkylsulfonylamino is a straight-chain or branched alkylsulfonylamino radical having 1 to 4, preferably 1 to 3, carbon atoms. Nonlimiting examples include methylsulfonylamino, ethylsulfonylamino, n-propylsulfonylamino, isopropyl-sulfonylamino, tert-butylsulfonylamino.Arylsulfonylamino is a naphthyl- or phenylsulfonylamino radical and preferably a phenylsulfonylamino radical.C1-C6-Alkylthio is a straight-chain or branched alkylthio radical having 1 to 6 carbon atoms. Preference is given to a straight-chain or branched alkylthio radical having 1 to 4, particularly preferably 1 to 3, carbon atoms. The following radicals may be mentioned by way of example and by way of preference: methylthio, ethylthio, n-propylthio, isopropylthio, tert-butylthio, n-pentylthio and n-hexylthio.Arylcarbonyl is a naphthyl- or phenylcarbonyl radical and preferably a phenylcarbonyl radical (Benzoyl radical).Heteroarylcarbonyl is a heteroarylcarbonyl radical having a 5- to 6-membered, preferably a 5-membered, heteroaryl ring having up to 2 heteroatoms selected from the group consisting of O, S and N. Nonlimiting examples include thienylcarbonyl, furylcarbonyl, pyrrolylcarbonyl, thiazolylcarbonyl, oxazolylcarbonyl, imidazolylcarbonyl, pyridylcarbonyl, pyrimidylcarbonyl.C3-C6-Cycloalkylcarbonylmethyl is a monocyclic cycloalkyl group having 3 to 6 carbon atoms which is attached via a carbonylmethyl group [—C(═O)—CH2—]. Nonlimiting examples include cyclopropylcarbonylmethyl, cyclopentylcarbonylmethyl and cyclohexylcarbonylmethyl.Halogen is fluorine, chlorine, bromine and iodine. Preference is given to fluorine, chlorine and bromine. Particular preference is given to fluorine and chlorine.
When radicals in the compounds according to the invention are optionally substituted, unless indicated otherwise the radicals may have one or more identical or different substituents. Preference is given to radicals substituted by up to three identical or different substituents.
Preference is given to compounds of the formula (I),
in which
    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen or (C1-C6)-alkyl,    R3 represents hydrogen, halogen or (C1-C6)-alkyl,    A represents oxygen or sulfur,and    the ring B represents benzo, pyrido, pyrimido, pyridazo or pyridazino which are optionally substituted by radicals selected from the group consisting of hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, (C1-C6)-alkyl, (C1-C6)-alkoxy, (C1-C6)-alkylthio and benzo.
Particular preference is given to compounds of the formula (I),
in which
    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen,    R3 represents hydrogen, chlorine, fluorine or methyl,    A represents oxygen or sulfur,and    the ring B represents benzo or pyrido, where benzo or pyrido is optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, C1-C4-alkyl, C1-C4-alkoxy, C1-C4-alkylthio, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl, 1,3-dioxapropane-1,3-diyl, amino(hydroxyimino)methyl and benzo.
Especially preferred are compounds of the formula (Ia)
in which    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen or C1-C6-alkyl,    R3 represents hydrogen, halogen or C1-C6-alkyl,    A represents oxygen or sulfur,and    Z represents hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, C1-C6-alkoxy, C1-C6-alkylthio, C1-C6-alkylamino, heteroarylcarbonylamino, arylcarbonylamino, C1-C4-alkylsulfonylamino, di(arylsulfonyl)amino, C3-C6-cycloalkylcarbonylmethyl or amino(hydroxyimino)methyl.
Very particular preference is given to compounds of the formula (Ia),
in which
    R1 represents 1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen,    R3 represents hydrogen, chlorine, fluorine or methyl,    A represents oxygen or sulfur,and    Z represents hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, methyl, ethyl, methoxy, ethoxy, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl or amino(hydroxyimino)methyl.
Particularly preferred are compounds of the formula (Ia),
in which
    R1 represents (3R)-1-azabicyclo[2.2.2]oct-3-yl,    R2 represents hydrogen,    R3 represents hydrogen, chlorine, fluorine or methyl,    A represents oxygen or sulfur,and    Z represents hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, methyl, ethyl, methoxy, ethoxy, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl or amino(hydroxyimino)methyl.
Also preferred are compounds of the formula (I),
in which
R1 represents (3R)-1-azabicyclo[2.2.2]oct-3-yl,
and R2, R3, A and the ring B are as defined above.
Particularly preferred are compounds of the formula (Ia),
in which
R1 represents (3R)-1-azabicyclo[2.2.2]oct-3-yl,
and R2, R3, A and Z are as defined above.
Also preferred are compounds of the formula (I),
in which
R2 represents hydrogen or methyl,
and R1, R3, A and the ring B are as defined above.
Particularly preferred are compounds of the formula (I),
in which
R2 represents hydrogen,
and R1, R3, A and the ring B are as defined above.
Also preferred are compounds of the formula (I),
in which
R3 represents hydrogen, fluorine, chlorine or methyl,
and R1, R2, A and the ring B are as defined above.
Particularly preferred are compounds of the formula (I),
in which
R3 represents hydrogen or methyl,
and R1, R2, A and the ring B are as defined above.
Very particularly preferred are compounds of the formula (I),
in which
R3 represents hydrogen,
and R1, R2, A and the ring B are as defined above.
Also preferred are compounds of the formula (I),
in which
A represents sulfur,
and R1, R2, R3 and the ring B are as defined above.
Also preferred are compounds of the formula (I),
in which
A represents oxygen,
and R1, R2, R3 and the ring B are as defined above.
Particularly preferred are compounds of the formula (Ia),
in which
A represents sulfur,
and R1, R2, R3 and Z are as defined above.
Also particularly preferred are compounds of the formula (Ia),
in which
A represents oxygen,
and R1, R2, R3 and Z are as defined above.
Also preferred are compounds of the formula (I),
in which
    the ring B represents benzo or pyrido, where benzo and pyrido are optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, C1-C4-alkyl, C1-C4-alkoxy, C1-C4-alkylthio, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl and amino(hydroxyimino)methyl,and R1, R2, R3 and A are as defined above.
Particularly preferred are compounds of the formula (I),
in which
the ring B represents benzo or pyrido, where benzo and pyrido are optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido and (C1-C4)-alkyl,and R1, R2, R3 and A are as defined above.
Very particular preference is given to compounds of formula (I),
in which
    the ring B represents benzo, where benzo is optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido and (C1-C4)-alkyl,and R1, R2, R3 and A are as defined above.
Also particularly preferred are compounds of the formula (Ia),
in which
    Z represents hydrogen, halogen, formyl, carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido, methyl, ethyl, methoxy, ethoxy, C1-C4-alkylamino, furylcarbonylamino, phenylcarbonylamino, methylsulfonylamino, di(phenylsulfonyl)amino, cyclopropylcarbonylmethyl or amino(hydroxyimino)methyl,and R1, R2, R3 and A are as defined above.
Very particular preference is given to combinations of two or more of the preferred ranges mentioned above.
Especially preferred are compounds of the formula (I),
in which
    R1 represents (3R)-1-azabicyclo[2.2.2]oct-3-yl,    R2 and R3 represent hydrogen,    A represents sulfur,and    the ring B represents benzo or pyrido, where benzo and pyrido are optionally substituted by 1 to 3 radicals selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, trifluoromethoxy, nitro, amino, formamido, acetamido and (C1-C4)-alkyl.
The invention relates furthermore to a process for preparing the compounds of the formula (I), characterized in that compounds of the formula (II)R1R2NH  (II),in which R1 and R2 are as defined aboveare reacted with a compound of the formula (III)
in whichR3, A and the ring B are as defined above, andX represents hydroxy or a suitable leaving group,in an inert solvent, if appropriate in the presence of a condensing agent and if appropriate in the presence of a base.
If X represents a leaving group, preference is given to chlorine, mesyloxy and isobutyloxycarbonyloxy, in particular to chlorine.
Inert solvents are, for example, halogenated hydrocarbons, such as methylene chloride, trichloromethane, carbon tetrachloride, trichloroethane, tetrachloroethane, 1,2-dichloroethane or trichloroethylene, ethers, such as diethyl ether, methyl tert-butyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether or diethylene glycol dimethyl ether, hydrocarbons, such as benzene, xylene, toluene, hexane, cyclohexane or mineral oil fractions, or other solvents, such as nitromethane, ethyl acetate, acetone, dimethylformamide, dimethylacetamide, 1,2-dimethoxyethane, dimethyl sulfoxide, acetonitrile or pyridine; preference is given to tetrahydrofuran, dimethylformamide or chloroform.
Condensing agents are, for example, carbodiimides, such as, for example, N,N′-diethyl-, N,N,′-dipropyl-, N,N′-diisopropyl-, N,N′-dicyclohexylcarbodiimide, N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-cyclohexylcarbodiimide-N′-propyloxymethyl-polystyrene (PS-carbodiimide), or carbonyl compounds, such as carbonyldiimidazole, or 1,2-oxazolium compounds, such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulfate or 2-tert-butyl-5-methylisoxazolium perchlorate, or acylamino compounds, such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or propanephosphonic anhydride, or isobutyl chloroformate, or bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride or benzotriazolyloxytri(dimethylamino)-phosphonium hexafluorophosphate, or O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), 2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU) or O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) or benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), or mixtures of these.
If appropriate, it may be advantageous to use these condensing agents in the presence of an auxiliary nucleophile, such as, for example, 1-hydroxybenzotriazole (HOBt).
Bases are, for example, alkali metal carbonates, such as, for example, sodium carbonate or potassium carbonate or sodium bicarbonate or potassium bicarbonate, or organic bases, such as trialkylamines, for example triethylamine, N-methylmorpholine, N-methylpiperidine, 4-N,N-dimethylaminopyridine or N,N-diisopropylethylamine.
Preference is given to O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) in the presence of N,N-diisopropylethylamine and to the combination of N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt), in each case in dimethylformamide.
The process according to the invention is preferably carried out in a temperature range of from room temperature to 50° C., at atmospheric pressure.
The compounds of the formulae (II) and (III) are known or can be synthesized by known processes from the appropriate starting materials (cf., for example, “Comprehensive Heterocyclic Chemistry”, Katritzki et al., Ed.; Elsevier, 1996).
Thus, for example, substituted benzothiophene-2-carboxylic acids can be obtained from appropriately substituted 2-halobenzaldehydes by reaction with methyl mercaptoacetate (see, for example, A. J. Bridges, A. Lee, E. C. Maduakor, Schwartz, Tetrahedron Lett. 1992, 33, 7499) and subsequent hydrolysis of the ester (synthesis scheme 1):

To synthesize the corresponding pyrido derivatives, it is possible to react 2-halo-benzonitrile starting materials with methyl mercaptoacetate to give the 3-aminobenzothiophene-2-carboxylic esters (synthesis scheme 2). The amino function can be removed by diazotization. Finally, the ester is hydrolyzed to give the target compound:

Substituted benzofuran-2-carboxylic acids can be obtained, for example, in accordance with D. Bogdal, M. Warzala, Tetrahedron 2000, 56, 8769 (synthesis scheme 3):

The compounds according to the invention of the formula (I) are suitable for use as medicaments for the treatment and/or prophylaxis of diseases in humans and/or animals.
The compounds according to the invention have an unforeseeable, useful pharmacological activity spectrum.
They act as ligands, in particular as α7-nAChR agonists.
The compounds of the invention can, because of their pharmacological properties, be employed alone or in combination with other active ingredients for the treatment and/or prevention of cognitive impairments, especially of Alzheimer's disease. Because of their selective effect as α7-nAChR agonists, the compounds of the invention are particularly suitable for improving perception, concentration, learning or memory, especially after cognitive impairments like those occurring for example in situations/diseases/syndromes such as mild cognitive impairment, age-associated learning and memory impairments, age-associated memory loss, vascular dementia, craniocerebral trauma, stroke, dementia occurring after strokes (post-stroke dementia), post-traumatic brain syndrome, general concentration impairments, concentration impairments in children with learning and memory problems, attention deficit hyperactivity disorder, Alzheimer's disease, Lewy body dementia, dementia with degeneration of the frontal lobes, including Pick's syndrome, Parkinson's disease, progressive nuclear palsy, dementia with corticobasal degeneration, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple sclerosis, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia, schizophrenia with dementia or Korsakoffs psychosis.
The compounds of the invention can be employed alone or in combination with other active ingredients for the prophylaxis and treatment of acute and/or chronic pain (for a classification, see “Classification of Chronic Pain, Descriptions of Chronic Pain Syndromes and Definitions of Pain Terms”, 2nd edition, Meskey and Begduk, editors; IASP Press, Seattle, 1994), especially for the treatment of cancer-induced pain and chronic neuropathic pain like, for example, that associated with diabetic neuropathy, postherpetic neuralgia, peripheral nerve damage, central pain (for example as a consequence of cerebral ischaemia) and trigeminal neuralgia, and other chronic pain such as, for example, lumbago, backache (low back pain) or rheumatic pain. In addition, these active ingredients are also suitable for the therapy of primary acute pain of any origin and of secondary states of pain resulting therefrom, and for the therapy of states of pain which were formerly acute and have become chronic.
The in vitro effect of the compounds of the invention can be shown in the following assays:
1. Determination of the Affinity of Test Substances for α7-nAChR by inhibition of [3H]-methyllycaconitine Binding to Rat Brain Membranes
The [3H]-methyllycaconitine binding assay is a modification of the method described by Davies et al. in Neuropharmacol. 1999, 38, 679-690.
Rat brain tissue (hippocampus or whole brain) is homogenized in homogenization buffer (10% w/v, 0.32 M sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.01% (w/v) NaN3, pH 7.4, 4° C.) at 600 rpm in a glass homogenizer. The homogenate is centrifuged (1000×g, 4° C., 10 min) and the supernatant is removed. The pellet is resuspended (20% w/v) and the suspension is centrifuged (1000×g, 4° C., 10 min). The two supernatants are combined and centrifuged (15 000×g, 4° C., 30 min). The pellet obtained in this way is referred to as the P2 fraction.
The P2 pellet is washed with binding buffer (50 mM Tris-HCl, 1 mM MgCl2, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, pH 7.4), and centrifuged (15 000×g, 4° C., 30 min), twice.
The P2 membranes are resuspended in binding buffer and incubated in a volume of 250 μl (amount of membrane protein 0.1-0.5 mg) in the presence of 1-5 nM [3H]-methyllycaconitine, 0.1% (w/v) BSA (bovine serum albumin) and various concentrations of the test substance at 21° C. for 2.5 h. Incubation is then carried out in the presence of 1 μM α-bungarotoxin or 100 μM nicotine or 10 μM MLA (methyllycaconitine) to determine the non-specific binding.
The incubation is stopped by adding 4 ml PBS (20 mM Na2HPO4, 5 mM KH2PO4, 150 mM NaCl, pH 7.4, 4° C.) and filtering through type A/E glass fibre filters (Gelman Sciences) which have previously been placed in 0.3% (v/v) polyethyleneimine (PEI) for 3 h. The filters are washed twice with 4 ml of PBS (4° C.), and the bound radioactivity is determined by scintillation measurement. All the assays are carried out in triplicate. The dissociation constant of the test substance Ki was determined from the IC50 of the compounds (concentration of the test substance at which 50% of the ligand bound to the receptor is displaced), the dissociation constant KD and the concentration L of [3H]-methyllycaconitine using the equation Ki=IC50/(1+L/KD).
In place of [3H]-methyllycaconitine it is also possible to employ other α7-nAChR-selective radioligands such as, for example, [125I]-α-bungarotoxin or nonselective nAChR radioligands together with inhibitors of other nAChRs.
Representative in vitro data for the effects of the compounds of the invention are shown in Table A:
TABLE AExampleKi (nM)1942.0353.73663.03780.04237.04658.04875.0513.9583.15920.06250.06420.06555.0682.0732.87422.0766.77720.07880.08031.08828.0
The suitability of the compounds of the invention for the treatment of cognitive impairments can be shown in the following animal models:
2. Object Recognition Test
The object recognition test is a memory test. It measures the ability of rats (and mice) to distinguish between familiar and unfamiliar objects.
The test is carried out as described by Blokland et al., NeuroReport 1998, 9, 4205-4208; A. Ennaceur J. Delacour, Behav. Brain Res. 1988, 31, 47-59; A. Ennaceur K. Meliani, Psychopharmacology 1992, 109, 321-330; and Prickaerts et al., Eur. J. Pharmacol. 1997, 337, 125-136.
In a first run, a rat is confronted in an otherwise empty observation arena of relatively large size by two identical objects. The rat will investigate, i.e. sniff round and touch, both objects extensively. In a second run, after an interval of 24 hours, the rat is put in the observation arena again. One of the familiar objects has now been replaced by a new, unfamiliar object. If a rat recognizes the familiar object, it will concentrate on investigating the unfamiliar object. However, after 24 hours, a rat has normally forgotten which object it investigated in the first run, and it will therefore inspect both objects to the same extent. Administration of a substance with a learning- and memory-improving effect may lead to a rat recognizing the object seen in the first run 24 hours previously as familiar. It will investigate the new, unfamiliar object in more detail than the familiar one. This memory ability is expressed in a discrimination index. A discrimination index of zero means that the rat investigates both objects, the old and the new, for equal times; that is to say it has not recognized the old object and reacts to both objects as if they were new and unfamiliar. A discrimination index greater than zero means that the rat inspects the new object for longer than the old one; that is to say the rat has recognized the old object.
3. Social Recognition Test:
The social recognition test is a test to examine the learning- or memory-improving effect of test substances.
Adult rats housed in groups are placed singly in test cages 30 minutes before the start of the test. Four minutes before the start of the test, the test animal is put in an observation box. After this adaptation time, a juvenile animal is put in with the test animal and the time for which the adult animal investigates the juvenile animal is measured for 2 minutes (trial 1). All behaviours clearly directed at the young animal are measured, i.e. anogenital inspection, pursuit and fur care, during which the old animal is no further than 1 cm from the young animal. The juvenile animal is then taken out, and the adult is left in its test cage (for 24-hour retention, the animal is returned to its home cage). The adult test animal is treated with test substance before or after the first test. Depending on the timing of the treatment, the learning or the storage of the information about the young animal can be influenced by the substance. After a fixed period (retention), the test is repeated (trial 2). A larger difference between the investigation times measured in trials 1 and 2 means that the adult animal has remembered the young animal better.
The compounds of the formula (I) according to the invention are suitable for use as medicaments for humans and animals.
The present invention also includes pharmaceutical preparations which, besides inert, nontoxic, pharmaceutically suitable excipients and carriers, contain one or more compounds of the formula (I), or which consist of one or more compounds of the formula (I), and processes for producing these preparations.
The compounds of the formula (I) are to be present in these preparations in a concentration of from 0.1 to 99.5% by weight, preferably from 0.5 to 95% by weight, of the complete mixture.
Besides the compounds of the formula (I), the pharmaceutical preparations may also contain other active pharmaceutical ingredients.
The abovementioned pharmaceutical preparations can be produced by known methods in a conventional way using, for example, the auxiliary or auxiliaries or excipient(s).
The novel active ingredients can be converted in a known manner into conventional formulations such as tablets, coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, nontoxic, pharmaceutically suitable excipients or solvents. In these cases, the therapeutically active compound should in each case be present in a concentration of about 0.5 to 90% by weight of the entire mixture, i.e. in amounts which are sufficient to reach the stated dose range.
The formulations are produced for example by extending the active ingredients with solvents and/or excipients, where appropriate with use of emulsifiers and/or dispersants, it being possible for example when water is used as diluent where appropriate to use organic solvents as auxiliary solvents.
Administration takes place in a conventional way, preferably orally, transdermally or parenterally, especially perlingually or intravenously. However, it can also take place by inhalation through the mouth or nose, for example with the aid of a spray, or topically via the skin.
It has generally proved advantageous to administer amounts of about 0.001 to 10 mg/kg, on oral administration preferably about 0.005 to 3 mg/kg, of body weight to achieve effective results.
It may, nevertheless, be necessary where appropriate to deviate from the stated amounts, in particular as a function of the body weight or of the mode of administration, of the individual behaviour toward the medicament, the nature of its formulation and the time or interval over which administration takes place. Thus, it may be sufficient in some cases to make do with less than the aforementioned minimum amount, whereas in other cases the stated upper limit must be exceeded. Where larger amounts are administered, it may be advisable to divide these into a plurality of single doses over the day.
Abbreviations:BINAP2,2′-Bis(diphenylphosphino)-1,1′-binaphthylDCIdirect chemical ionization (in MS)DMFN,N-DimethylformamideDMSODimethyl sulfoxideEDCN′-(3-Dimethylaminopropyl)-N-ethylcarbodiimide × HClEDTAEthylenediaminetetraacetic acideq.Equivalent(s)ESIElectrospray ionization (in MS)HATUO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphateHOBt1-Hydroxy-1H-benzotriazole × H2OHPLCHigh pressure/high performance liquid chromatographyLC-MSLiquid chromatography with coupled mass spectroscopyMSMass spectroscopyNMRNuclear magnetic resonance spectroscopyPd2(dba)3Tris(dibenzylideneacetone)dipalladium(0)RTRoom temperature, 20° C.RtRetention time (in HPLC)TBTUO-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborateTFATrifluoroacetic acidTHFTetrahydrofuranTrisTris(hydroxymethyl)aminomethaneLC-MS Method A:
MS instrument:Micromass Quattro LCZIonization: ESI positiveHPLC instrument:HP 1100UV detector DAD: 208-400 nmOven temperature: 40° C.Column:Symmetry C 1850 mm × 2.1 mm; 3.5 μmGradient:TimeA:B:Flow rate(min)%%(ml/min)0.0010.090.00.504.0090.010.00.506.0090.010.00.506.1010.090.01.007.5010.090.00.50A: Acetonitrile + 0.1% formic acidB: Water + 0.1% formic acidLC-MS Method B:
MS instrument:Finnigan MAT 900SIonization: ESI positiveHPLC instrument:TSP: P4000, AS3000, UV3000HRUV detector 3000HR: 210 nmOven temperature: 70° C.Column:Symmetry C 18 150 mm × 2.1 mm; 5 μmSupplier:WatersTimeFlow rate(min)A: %B: %C: %(ml/min)Gradient:0 249490.92.595 2.5 2.51.2595 2.5 2.51.25.5 249491.26.5 249491.27 249490.9A: AcetonitrileB: Water + 0.6 g/l 35% strength hydrochloric acidC: WaterLC-MS Method C:
MS instrument:Micromass Platform LCZIonization: ESI positiveHPLC instrument:HP 1100UV detector DAD: 208-400 nmOven temperature: 40° C.Column:Symmetry C 1850 mm × 2.1 mm; 3.5 μmGradientTimeA:B:Flow rate(min)%%(ml/min)0.0010.090.00.504.0090.010.00.506.0090.010.00.506.1010.090.01.007.5010.090.00.50A: Acetonitrile + 0.1% formic acidB: Water + 0.1% formic acidLC-MS Method D:
Instrument: Micromass Platform LCZ, HP1100; Column: Symmetry C18, 50 mm×2.1 mm, 3.5 μm; Mobile phase A: water+0.05% formic acid, mobile phase B: acetonitrile+0.05% formic acid; Gradient: 0.0 min 90% A→4.0 min 10% A→6.0 min 10% A; Oven: 40° C.; Flow rate: 0.5 ml/min; UV detection: 208-400 nm.
LC-MS Method E:
Instrument: Micromass Quattro LCZ, HP1100; Column: Uptisphere HDO, 50 mm×2.0 mm, 3 μm; Mobile phase A: water+0.05% formic acid, mobile phase B: acetonitrile+0.05% formic acid; Gradient: 0.0 min 100% A→0.2 min 100% A→2.9 min 30% A→3.1 min 10% A→4.5 min 10% A; Oven: 55° C.; Flow rate: 0.8 ml/min; UV detection: 208-400 nm.
LC-MS Method F:
Instrument: Micromass Quattro LCZ, HP1100; Column: Symmetry C18, 50 mm×2.1 mm, 3.5 μm; Mobile phase A: water+0.05% formic acid, mobile phase B: acetonitrile+0.05% formic acid; Gradient: 0.0 min 90% A→4.0 min 10% A→6.0 min 10% A; Oven: 40° C.; Flow rate: 0.5 ml/min; UV detection: 208-400 nm.
LC-MS Method G:
MS instrument: Micromass ZQ; HPLC instrument: Waters Alliance 2790; Column: Uptisphere C18, 50 mm×2.0 mm, 3.0 μm; Mobile phase A: acetonitrile+0.05% formic acid, mobile phase B: water+0.05% formic acid; Gradient: 0.0 min 5% A→2.0 min 40% A→4.5 min 90% A→5.5 min 90% A; Oven: 45° C.; Flow rate: 0.0 min 0.75 ml/min→4.5 min 0.75 ml/min→5.5 min 1.25 ml/min; UV detection: 210 nm.
HPLC Method H:
Instrument: HP 1100 with DAD detection; column: Kromasil RP-18, 60 mm×2 mm, 3.5 μm; mobile phase A: 5 ml HClO4/l H2O, mobile phase B: acetonitrile; Gradient: 0 min 2% B, 0.5 min 2% B, 4.5 min 90% B, 6.5 min 90% B; Flow rate: 0.75 ml/min; Temperature: 30° C.; UV detection: 210 nm.
Starting Materials:
General reaction scheme for the synthesis of methyl 1-benzothiophene-2-carboxylates:
General Procedure for the Synthesis of methyl 1-benzothiophene-2-carboxylates:
Under an argon atmosphere, 1.5 equivalents of sodium hydride (60% pure) are initially charged in absolute DMSO (0.60-1.26 M suspension). At room temperature, 1.1 equivalents of methyl mercaptoacetate are slowly added dropwise to the reaction mixture, and it is left to stir at room temperature until evolution of hydrogen ceases (about 15 min). 1.0 equivalents of the appropriate benzaldehyde are dissolved in absolute DMSO (1.60-3.36 M solution) and added at room temperature to the reaction mixture. The reaction mixture is stirred until the reaction is complete (about 5-10 min) and then poured into ice-water. The resulting precipitate is filtered off with suction, dried at 40° C. under reduced pressure overnight and reacted further as crude product.
General Reaction Scheme for the Synthesis of 1-benzothiophene-2-carboxylic acids:
General Procedure for the Synthesis of 1-benzothiophene-2-carboxylic acids:
A mixture of equal parts of THF and 2 N potassium hydroxide solution (0.28-0.47 M solution) is added to the appropriate methyl 1-benzothiophene-2-carboxylate. The reaction mixture is left to stir at room temperature overnight. The THF is removed under reduced pressure and the aqueous reaction mixture is acidified with concentrated hydrochloric acid. The resulting precipitate is filtered off with suction and dried under reduced pressure at 40° C.
General Procedure for Amide Linkage Between 3-quinuclidinamine and 2-benzothiophene- or 2-benzofurancarboxylic acids (Variant A):
1.5 eq. of the appropriate enantiomeric 3-quinuclidinamine hydrochloride are, together with 1 eq. of the carboxylic acid and 1.5 eq. of HATU, initially charged in DMF at 0° C. After addition of 1.5 eq. of N,N-diisopropylethylamine, the mixture is stirred for 30 min. A further 4 eq. of N,N-diisopropylethylamine are added, and the mixture is stirred at RT overnight. Purification is carried out chromatographically.
General Procedure for Amide Linkage Between 3-quinuclidinamine and 2-benzothiophene- or 2-benzofurancarboxylic acids (Variant B):
1.0 eq. of the appropriate enantiomeric 3-quinuclidinamine dihydrochloride are, together with 1 eq. of the carboxylic acid and 1.2 eq. of HATU, initially charged in DMF at 0° C. After addition of 1.2 eq. of N,N-diisopropylethylamine, the mixture is stirred for 30 min. A further 2.4 eq. of N,N-diisopropylethylamine are added, and the mixture is stirred at RT overnight. Purification is carried out chromatographically.
General Procedure for the Synthesis of methyl 3-aminothienopyridine-2-carboxylates:
1.0 equivalents of the appropriate pyridine derivative are dissolved in absolute DMSO (0.93-0.96 M solution), and 2 equivalents of triethylamine are added. After addition of 1 equivalent of methyl mercaptoacetate, the reaction mixture is stirred at 60° C. overnight. The reaction mixture is poured into ice-water and stirred therein. The precipitated solid is filtered off with suction and, if required, purified by column chromatography (silica gel 60, mobile phase toluene/ethyl acetate 20:1 to 5:1).
General Procedure for the Synthesis of methyl thienopyridine-2-carboxylates:
With cooling at −5° C., 1.0 equivalents of the appropriate methyl 3-aminothienopyridine-2-carboxylate is initially charged in 75% strength sulfuric acid (0.33-0.36 M solution). A solution of 3.2 equivalents of sodium nitrite in water (0.92-1.00 M solution) is slowly added dropwise to the reaction mixture such that the temperature of the reaction mixture does not exceed 0° C. The mixture is stirred at 0° C. for 45 min. 60-65 equivalents of ice-cold 50% strength hypophosphoric acid are then added dropwise to the reaction mixture, again in such a way that the temperature does not exceed 0° C. The reaction mixture is stirred at −5° C. for one hour and then kept in the fridge overnight. The reaction mixture is diluted with sodium bicarbonate solution and ethyl acetate, and sodium bicarbonate is added a little at a time until the mixture gives a basic reaction. Work-up is described in the examples below.
General Procedure for the Synthesis of thienopyridine-2-carboxylic acids:
A mixture of equal parts of THF and 2 N potassium hydroxide solution (0.22 M solution) is added to the appropriate methyl thienopyridine-2-carboxylate. The reaction mixture is stirred at room temperature overnight. The reaction mixture is diluted with water, washed twice with ethyl acetate and acidified with concentrated hydrochloric acid. Work-up is described in the examples below.